Each year, Mycobacterium tuberculosis (Mtb) infection causes 1.8 million deaths worldwide. The inadequacies of present tuberculosis (TB) therapies demand the discovery of new agents to treat Mtb infection. In prior work, we have identified CarD as a transcriptional regulator that is necessary for Mtb pathogenesis, contributes to rifampicin resistance, regulates ribosomal RNA (rRNA) levels, and is not present in eukaryotes. CarD is thus an attractive drug target, but knowledge of the molecular details of CarD function is required to develop specific inhibitors of CarD activity. We hypothesize that since CarD is required for regulating transcription, then its structural domains perform specific functions during transcription and their activity can be inhibited to compromise these processes. We will utilize an innovative single-molecule approach to monitor transcription by mycobacterial RNA polymerase (RNAP) from mycobacterial rRNA promoters in real time and determine how CarD modulates each individual phase of transcription. Specifically, the following aims will address the mechanism of CarD at the molecular, biochemical, and biophysical levels to gain insight into Mtb pathogenesis and to expand paradigms of prokaryotic transcription. Aim 1. Elucidate the mechanism of action of CarD at rRNA promoters. We will use single molecule techniques to quantitatively determine the effect of CarD on different stages of transcription and learn how CarD affects transcription kinetics. Aim 2. Determine the effect of CarD on rifampicin sensitivity of RNAP. We will measure the effect of CarD on the detailed kinetics of transcription initiation and abortive transcription in the presence of rifampicin. Aim 3. Investigate the role o CarD macromolecular interactions during transcription regulation. Using point mutations in CarD, we will determine how disruptions in the macromolecular interactions between CarD, RNAP, and the promoter affect CarD regulation of transcription and rifampicin resistance. The outcome of this work will be a detailed mechanism of CarD activity, which will provide answers to fundamental questions regarding transcription regulation in mycobacteria. Our investigations will generate insight into the essential activity of CarD that may then be targeted in new chemotherapeutic strategies to treat TB. Notably, CarD is conserved in many other bacteria, indicating that our findings will apply to diverse bacterial pathogens. Thus, the proposed research will advance the mission of the National Institutes of Health to gain fundamental knowledge to decrease the burden of infectious disease on human health.